The objective of this proposal is to characterize the distinctive signaling pathways that initiate, sustain, and terminate contraction of longitudinal colonic smooth muscle and identify the changes in expression and activity of specific targets that determine hypercontractility of this muscle during inflammation. Contraction in longitudinal muscle is initiated by cPLA2-dependent Ca2+ influx leading to Ca2+- and cyclic ADP ribose-induced Ca2+ release via ryanodine (RYR2) receptors. Preliminary studies showed that initial contraction was terminated by inactivation of MLCK via a novel pathway involving sequential activation of CaMK kinase and AMPK. Preliminary studies also showed that sustained contraction was mediated via sequential activation of G12, LARG, RhoA/Rho kinase leading to inhibition of MLC phosphatase. The pathways for relaxation via PKA and PKG in longitudinal muscle were also distinct and involved inhibitory phosphorylation of cPLA2 and ADP ribosyl cyclase during initial contraction and stimulatory phosphorylation of the MLCP activator, telokin, during sustained contraction. Preliminary studies in IL-1?-treated colonic longitudinal muscle strips in vitro and muscle strips from TNBS-colitis in vivo showed similar changes in the expression and/or activity of specific signaling targets that resulted in hypercontractility during inflammation. These targets included: (i) s-nitrosylation and activation of RYR2 via iNOS, leading to increase in Ca2+ release, and NF-?B/PKA-mediated inactivation of AMPK, leading to increase in MLCK activity, MLC20 phosphorylation, and initial contraction;and (ii) up-regulation of LARG via an iNOS/JNK/AP-1 pathway, and down-regulation of telokin via an NF-?B pathway, leading to further inhibition of MLCP and increase in sustained MLC20 phosphorylation and contraction. Preliminary studies also showed that relaxation mediated by PKA and PKG in longitudinal muscle is attenuated during inflammation. The targets included: (i) s-nitrosylation of sGC and ACIII leading to inhibition of cGMP and cAMP synthesis, and (ii) up-regulation of PDE1A1 via NF-?B, and ERK1/2- mediated activation of PDE4D5, leading to augmentation of cGMP and cAMP degradation. The specific aims are to characterize novel signaling pathways that initiate, sustain, and terminate contraction in colonic longitudinal muscle, identify the specific targets whose expression and/or activity is altered during inflammation, and determine their regulation via iNOS/JNK/AP-1, NF-:B, and ERK1/2. These targets include RYR2, AMPK, LARG, and telokin (Aims I and II);and sGC, ACIII, PDE1A1, and PDE4D5 (Aim III). The innovative aspects of this proposal lie in the discovery of mechanisms of hypercontractility of longitudinal muscle during inflammation. PUBLIC HEALTH RELEVANCE: The objective of this proposal is to characterize the distinctive signaling pathways that initiate, sustain, and terminate contraction and relaxation of longitudinal intestinal smooth muscle and identify the changes in expression and activity of specific targets that determine hypercontractiliy of this muscle during inflammation. The project involves a comprehensive analysis of the mechanisms by which inflammatory cytokines augment contraction and inhibit relaxation at cellular level, both in vitro and in an established model of colonic inflammation.